JP2004163421A - Magnetic substance-including particle and its production method, and immunoassay particle using magnetic substance-including particle - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a magnetic substance-including particles useful for immunoassay, having uniform magnetism and excellent dispersion stability with a narrow particle size distribution, its production method, and immunoassay particles using it. <P>SOLUTION: This magnetic substance-including particles formed of an organic high molecular material and a magnetic substance having an average particle size of 1-30 nm. The magnetic substance is included in the inner part in a dispersed state. <P>COPYRIGHT: (C)2004,JPO

Description

  The present invention provides a polymer particle having a uniform magnetic property, a narrow particle size distribution and excellent dispersion stability, and encapsulating a magnetic material useful as an immunoassay particle, a method for producing the same, and a method for producing the same Related to immunoassay particles.

  Conventionally, magnetic body-encapsulated particles are produced by (1) a method of producing magnetic body-encapsulated particles by including iron ions in the prepared polymer particles, and (2) a process of polymerizing particles from monomers. (3) A method of combining separately prepared polymer particles and magnetic particles (see Patent Document 2) is known. In addition, there is (4) a method of coating magnetic particles with a polymer or the like (see Patent Document 3).

  In the method (1), since iron ions are absorbed by polymer particles, there is a problem that the magnetic material is exposed on the surface and the magnetic material is oxidized. The method (2) has a problem that the magnetic particles are not uniformly taken into the particles, and it is difficult to control the particle size, and there is a problem that the product has a wide particle size distribution. The method (3) has a problem that the polymer particles aggregate and cannot be used for particles having a small particle size. The method (4) has problems in that the coating cannot be made uniform, so that the floatability and dispersibility are poor, and a part of the surface of the magnetic particles may be exposed.

On the other hand, radioimmunoassay, enzyme immunoassay, fluorescent immunoassay and the like have already been put to practical use as conventionally known microimmunoassay methods. These methods are methods for detecting the presence or absence of an antibody or antigen that specifically reacts with an antigen or an antibody to which an isotope, enzyme, or fluorescent substance is added as a label, respectively.
In such an immunoassay, the magnetic particles are used to perform B / F separation efficiently and simply. In addition, uses other than B / F separation (see Patent Document 4) and immunoassay methods (see Patent Document 5, Patent Document 6, and Patent Document 7) using magnetic inclusion particles themselves as labeling materials are disclosed.

JP-A-9-208788 JP-A-6-231957 JP-A-6-92640 JP 2000-88852 A JP-A-6-148189 JP 7-225233 A JP 2001-524675 A

  In view of the above situation, the present invention has a uniform magnetic property, a narrow particle size distribution and excellent dispersion stability, and is useful for immunoassay, a method for producing the same, and a method for producing the same. It is an object to provide particles for immunoassay.

The present invention is a magnetic substance-encapsulated particle comprising an organic polymer substance and a magnetic substance having an average particle size of 1 to 30 nm, and containing the magnetic substance in a dispersed state therein.
The present invention is described in detail below.

The magnetic substance-encapsulated particles of the present invention are composed of an organic polymer substance and a magnetic substance having an average particle diameter of 1 to 30 nm.
The organic polymer material includes a hydrophobic monomer for forming a core of the polymer particle and / or a hydrophilic property for forming a polymer particle shell while forming the polymer particle stably dispersed in water. A polymer composed of monomers is used as a main constituent.
In particular, when the amount of the hydrophobic monomer in the organic polymer material is reduced, it becomes difficult to incorporate metal ions described later during particle polymerization, and when the amount of the hydrophilic monomer is reduced, the dispersion stability of the magnetic substance-encapsulated particles obtained is reduced. These are preferably used in combination, and the proportion is preferably 5 to 97% by weight for the hydrophobic monomer and 3 to 95% by weight for the hydrophilic monomer, and is adjusted as necessary within this range. More preferably.

<Hydrophobic monomer>
Examples of the hydrophobic monomer include styrene; styrene derivatives such as α-methylstyrene, p-methylstyrene, p-chlorostyrene, and chloromethylstyrene; vinyl chloride; vinyl esters such as vinyl acetate and vinyl propionate; acrylonitrile. Unsaturated nitriles such as: methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, stearyl (meth) acrylate, ethylene glycol (meth) acrylate , (Meth) acrylic acid ester derivatives such as trifluoroethyl (meth) acrylate, pentafluoropropyl (meth) acrylate, cyclohexyl (meth) acrylate, glycidyl methacrylate, tetrahydrofurfuryl (meth) acrylate And the like. These monomers may be used independently and may use 2 or more types together.

  The hydrophobic monomer is preferably methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, stearyl (meth) acrylate, ethylene glycol ( Acrylic monomers such as (meth) acrylate derivatives such as (meth) acrylate, trifluoroethyl (meth) acrylate, pentafluoropropyl (meth) acrylate, cyclohexyl (meth) acrylate, glycidyl methacrylate, and tetrahydrofurfuryl (meth) acrylate Is used.

  The hydrophobic monomer is more preferably an acryl having a glycidyl group having an excellent ability to take in metal ions at a high concentration during particle polymerization because particle formation by polymerization and magnetic substance formation proceed simultaneously as described later. System monomers are used. That is, among the acrylic monomers, glycidyl methacrylate (GMA) is particularly preferably used because of its high affinity with iron ions and magnetite.

A crosslinkable monomer can also be used as the hydrophobic monomer. Examples of the crosslinkable monomer include divinylbenzene, divinylbiphenyl, divinylnaphthalene, ethylene glycol di (meth) acrylate, 1,6-hexanediol di (meth) acrylate, neopentyl glycol di (meth) acrylate, Examples include methylolpropane tri (meth) acrylate, tetramethylolmethane tri (meth) acrylate, tetramethylolpropane tetra (meth) acrylate, diallyl phthalate and isomers thereof, triallyl isocyanurate and derivatives thereof. These crosslinkable monomers may be used independently and may use 2 or more types together.
Among these, ethylene glycol di (meth) acrylate is preferably used because of its high affinity with iron ions and magnetite.

<Hydrophilic monomer>
Examples of the hydrophilic monomer include carboxylic acids having a polymerizable unsaturated bond such as acrylic acid, methacrylic acid, itaconic acid, fumaric acid, and maleic acid; phosphate esters having a polymerizable unsaturated bond; Sulfonic acid ester having a bond; salt of an amine having an acryloyl group such as dimethylaminoethyl methacrylate quaternary salt or diethylaminoethyl methacrylate quaternary salt; a cationic group such as a salt of a nitrogen-containing compound having a vinyl group such as vinylpyridine Vinyl monomers: Nonionic vinyl monomers such as 2-hydroxyethyl methacrylate, polyethylene glycol (meth) acrylate, (meth) acrylamide, methylol acrylamide, and glycerol methacrylate (GLM). These hydrophilic monomers may be used independently and may use 2 or more types together. Among these, polyethylene glycol (meth) acrylate represented by the following general formula is preferably used because it has a high ability to stably disperse particles in water and does not hinder the formation of a magnetic substance.
_{CH 2 = CR-COO- (CH} 2 -CH 2 -O) n-H
In the formula, R represents H or CH ₃ , and n represents an integer of 2 to 20. The preferable lower limit of n is 2, and the preferable upper limit is 10.

The magnetic material is preferably formed by oxidation of metal ions inside the particles in the polymerization process for forming the particles.
<Metal ion>
Although the said metal ion will not be specifically limited if it forms a magnetic body, Preferably, they are an iron ion, cobalt ion, nickel ion, etc., More preferably, it is an iron ion. Magnetite, which is a magnetic substance, is obtained by oxidizing ferric chloride with an oxidizing agent or the like.

  The average particle diameter of the magnetic material is 1 to 30 nm. When the thickness is less than 1 nm, the magnetic response characteristics of the magnetic substance are reduced, and when used for immunoassay, the measurement sensitivity is lowered, making it difficult to perform qualitative and quantitative analysis. On the other hand, if the thickness exceeds 30 nm, the dispersibility in the magnetic substance-encapsulated particles decreases, and the magnetism becomes non-uniform. In this case also, it becomes difficult to perform qualitative and quantitative analysis when used for immunoassay. A preferred lower limit is 2 nm and a preferred upper limit is 20 nm. Further, the preferable upper limit is 10 nm, and it is usually difficult to form such fine magnetic particles, but as in the manufacturing method described later, while forming the particles and incorporating metal ions into the particles By simultaneously proceeding with the step of oxidizing the metal ions to form the magnetic body, it becomes possible to form a minute magnetic body.

  The magnetic substance-encapsulated particles of the present invention contain the above magnetic substance in a dispersed state. That is, in the magnetic substance-encapsulated particle of the present invention, the magnetic substance is present in a dispersed state inside the particle without being exposed on the particle surface.

  The magnetic substance content of the magnetic substance-encapsulated particles of the present invention is preferably adjusted in the range of 0.1 to 40% by weight depending on the polymerization composition. If it is less than 0.1% by weight, the magnetic response characteristics of the magnetic substance-encapsulated particles are reduced, and when used for immunoassay, the measurement sensitivity is lowered, and it becomes difficult to perform qualitative and quantitative analysis. On the other hand, if it exceeds 40% by weight, the polymerization operability of the particles is lowered, and it becomes difficult to incorporate metal ions during the particle polymerization. A preferred lower limit is 1% by weight and a preferred upper limit is 30% by weight.

  The average particle diameter of the magnetic substance-encapsulated particles of the present invention is preferably adjusted in the range of 0.05 to 1 μm depending on the polymerization conditions. If it is less than 0.05 μm or more than 1 μm, it becomes difficult to control the particle shape and polymerization of the magnetic particles, and if it is less than 0.05 μm, the sensitivity of the measurement decreases when applied to an immunoassay. Quantitative analysis becomes difficult, and when it exceeds 1 μm, when applied to an immunoassay, particles are likely to settle over time due to aggregation in the dispersion, and in this case also, qualitative and quantitative analysis becomes difficult. A preferable lower limit is 0.07 μm, and a preferable upper limit is 0.8 μm.

  The magnetic substance-encapsulated particles of the present invention include a step of polymerizing a hydrophobic monomer and / or a hydrophilic monomer in an aqueous solvent to form particles, and oxidizing the metal ions while incorporating the metal ions into the particles. And the step of forming the particles, and the step of simultaneously forming the step of forming the particles and the step of forming the magnetic body. Such a manufacturing method is also one aspect of the present invention.

In the step of forming particles by polymerizing a hydrophobic monomer and / or a hydrophilic monomer in an aqueous solvent, it is preferable to add a polymerization initiator.
<Polymerization initiator>
The polymerization initiator is not particularly limited, and examples thereof include water-soluble organic azo compounds, inorganic peroxides, and organic peroxides.
Preferable examples of the polymerization initiator include potassium persulfate (KPS; polymerization temperature 70 ° C.), azobisamidinopropane hydrochloride (V-50; polymerization temperature 70 ° C.), 2,2-azobis [2- (2 -Imidazolin-2-yl) propane] dihydrochloride (VA-044; polymerization temperature 60 ° C.) and the like. Among these, KPS, which is a peroxide polymerization initiator, is expected to contribute to the oxidation of divalent iron ions at the start of polymerization, and is assumed to proceed simultaneously with polymerization and magnetite formation by monomers and iron ions. doing. V-50 and VA-044 are weak in oxidizing power and become polymerization initiators involved in the mild oxidation reaction of divalent iron ions.
Since the polymerization initiator may lose its radical activity due to Fe ²⁺ oxidation or Fe ³⁺ , it is effective to add it later in the particle growth process for the purpose of promoting particle growth by polymerization. In this case, new secondary particles are not formed, and the particle surface is coated with the polymer.

<PH adjuster>
In the present invention, when forming a magnetic material simultaneously with polymerization, it is important to adjust the pH in the polymerization system to basic. For example, in a system using KPS as a polymerization initiator, dispersion stability in water is good as a merit, and monodisperse particles with a narrow particle size distribution can be obtained. May become acidic, and the resulting magnetic inclusion particles may become particles that are weakly attracted to the magnet. On the other hand, a merit in a system using VA-044 having no oxidizing power as a polymerization initiator is that the pH in the polymerization system is almost neutral.
In order to keep the pH in the polymerization system weakly basic, a general base can be used as a pH adjuster. Preferably NH ₄ OH is used as a pH adjuster.
The pH adjuster can be added several times as necessary.

<Polymerization method>
The magnetic body-encapsulated particles of the present invention can use particle polymerization methods such as suspension polymerization, dispersion polymerization, emulsion polymerization, soap-free emulsion polymerization, etc., but the Cv value of the obtained magnetic body-encapsulated particles should be 5% or less. Therefore, it is preferably produced by soap-free emulsion polymerization excellent in control of particle size distribution.
Hereinafter, although the manufacturing method of the magnetic body inclusion particle | grains by soap free emulsion polymerization is illustrated, it is not limited to this method.

A typical polymerization composition is as follows.
Monomer composition comprising hydrophilic monomer / hydrophobic monomer / reactive emulsifier: 3 g
H ₂ O: 100 g
The monomer composition and water are weighed into a four-necked flask. A stir bar and a reflux condenser are attached to each port. Next, in a system using KPS and V-50 as a polymerization initiator, a system using 70 ° C. and a system using VA-044 are placed in a thermostatic bath at 50 ° C. to 60 ° C., and the inside of the system is replaced with nitrogen while stirring. Thereafter, a polymerization initiator dissolved in water is injected into the system with a syringe. The polymerization is started at this time, and an aqueous solution of FeCl ₂ .4H ₂ O serving as a magnetic source is injected using a syringe after a predetermined time. FeCl ₂ .4H ₂ O is used by dissolving 1 to 3 to 4 moles of the polymerization initiator in 5 g of water. That is, a magnetic substance-encapsulated particle is produced by initiating polymerization of the above-described monomer with a polymerization initiator and oxidizing (magnetizing) divalent iron ions.
The polymerization is preferably performed for 2 to 24 hours from the start. In order to obtain an appropriate oxidizing power, NH ₄ OH may be added during the polymerization, and further, a polymerization initiator may be added during the polymerization in order to promote particle growth by the polymerization. In this way, magnetic body-encapsulated particles, which are polymer particles encapsulating a magnetic body, can be obtained.

The reactive emulsifier is a copolymerization monomer and may be added as necessary.
<Reactive emulsifier>
Examples of the reactive emulsifier include reactive emulsifiers represented by the following general formula, and these reactive emulsifiers may be used alone or in combination of two or more.

  The obtained magnetic substance-encapsulated particles are purified by repeated centrifugation and redispersion with distilled water in order to remove residual monomers, polymerization initiators, unreacted iron ions, and the like. After centrifugation, the supernatant is discarded by decantation, distilled water is added, and redispersion is performed with a glass rod. After purification, transfer to a glass vial, seal and store with a lid or parafilm.

The magnetic body-encapsulated particles of the present invention are very suitable for immunoassay, and the immunoassay particles can be obtained by adsorbing or binding an antigen or antibody to the magnetic body-encapsulated particles. Such immunoassay particles are also one aspect of the present invention.
The method for adsorbing or binding the antibody or antigen to the magnetic substance-encapsulated particle of the present invention is not particularly limited, and for example, a known method such as a physical adsorption method or a chemical bonding method using carbodiimide can be used.

The magnetic substance-encapsulated particle of the present invention or the immunoassay particle of the present invention can be suitably used for an immunoassay.
Examples of the immunoassay include known methods such as radioimmunoassay and enzyme immunoassay using magnetic inclusion particles as a carrier, and the target antigen or antibody can be measured by a sandwich method or a competitive method. it can. In addition, magnetic substance-encapsulated particles can be used as a label instead of isotopes, enzymes, and the like, which are labeling substances in the above method.

  Since the magnetic substance-encapsulated particle of the present invention is a particle having a narrow particle size distribution and containing a magnetic substance uniformly dispersed therein, the magnetic substance-encapsulated particle of the present invention is used to improve sensitivity in an immunoassay method using a magnetic substance as a label. It can be measured accurately. That is, immunoassay particles in which an antibody or antigen that specifically reacts with the antigen or antibody to be measured is bound to a magnetic substance-encapsulated particle are used, and this immunoassay particle reacts with the antigen or antibody to be measured. Then, the antigen or antibody to be measured can be qualitatively and quantitatively measured by measuring the magnetic content of the magnetic particles in the reacted immunoassay particles.

Since the magnetic substance-encapsulated particle of the invention according to claim 1 contains a magnetic substance having an average particle diameter of 1 to 30 nm inside, it is excellent in magnetic response characteristics of the magnetic substance and is contained in the magnetic substance-encapsulated particle. In addition, the magnetic substance-encapsulated particles have a uniform magnetism, excellent magnetic response characteristics, and a narrow particle size distribution and a uniform particle size. It is. Therefore, it can be used suitably for immunoassay that requires measurement sensitivity, etc., and by measuring the magnetism of magnetic inclusion particles that contributed to the immune reaction in the immunoassay method, it is qualitative with excellent measurement sensitivity. Not only can quantitative immunoassay be performed. Moreover, since the surface is formed from an organic polymer substance, an antigen or an antibody can be reliably bound.
The magnetic substance-encapsulated particles of the invention described in claim 2 and the magnetic substance-encapsulated particles of the invention described in claim 3 are formed by oxidizing a magnetic substance with metal ions, preferably iron ions inside the particles. Therefore, the magnetic substance is reliably and uniformly incorporated in the magnetic substance-encapsulated particles, and the measurement sensitivity required for the immunoassay can be reliably expressed.
In the magnetic body-encapsulated particle of the invention according to claim 4, since the organic polymer substance constituting the magnetic body-encapsulated particle is an acrylic monomer, the particle formation by the polymerization is surely performed, and the particle formation by the polymerization and the magnetic The body formation can surely proceed simultaneously. Therefore, the magnetic substance-encapsulated particles have a uniform magnetism and a narrow particle size distribution, and can exhibit better measurement sensitivity in an immunoassay.
In the magnetic body-encapsulated particle of the invention according to claim 5, since the organic polymer substance constituting the magnetic body-encapsulated particle is an acrylic monomer having a glycidyl group, iron ions and magnetite suitable for forming a magnetic body Therefore, in the magnetic substance-encapsulated particles, the magnetic substance is more uniformly dispersed, and the measurement sensitivity required for the immunoassay is more excellent.
In the magnetic body-encapsulated particle of the invention described in claim 6, since the organic polymer material constituting the magnetic body-encapsulated particle is a combination of a specific hydrophobic acrylic monomer and a hydrophilic acrylic monomer, Not only is it easy to take up metal ions uniformly, but it also excels in dispersion stability of the magnetic particles, so that the particles do not settle over time due to aggregation in the dispersion, and there is no need for sonication, etc. Therefore, it is possible to express excellent measurement sensitivity in an immunoassay method and the like.
The magnetic substance-encapsulated particles according to the seventh aspect of the present invention have an average particle diameter of 0.05 to 1 μm, so that the polymerization operation is easy, that is, the magnetic substance-encapsulated particles as described above can be obtained with certainty.
Since the magnetic substance-encapsulated particle according to the eighth aspect of the present invention has a magnetic substance content of 0.1 to 40% by weight, an excellent magnetic response characteristic can be expressed uniformly and reliably. It can apply more suitably. In addition, the polymerization operation is easy, and the above-mentioned magnetic substance-encapsulated particles can be obtained with certainty.
In the manufacturing method of the invention according to claims 9 to 13, since the step of forming the particles and the step of forming the magnetic substance are simultaneously advanced, the above-mentioned magnetic substance-encapsulated particles can be obtained easily and reliably. .
In the immunoassay particle according to the fourteenth aspect of the invention, since the magnetic inclusion particles as a carrier contain a magnetic substance having an average particle diameter of 1 to 30 nm, the magnetism is uniform and the magnetic response characteristics In addition, the particle size distribution is narrow and the particle size is uniform. In addition, since the surface is formed of an organic polymer material, antigens or antibodies are reliably bound. Therefore, it can be used suitably for immunoassays that require measurement sensitivity, etc., and by measuring the magnetism of the magnetic particles in the immunoassay particles that contributed to the immune reaction in the immunoassay, an excellent measurement Sensitivity enables quantitative immunoassay as well as qualitative.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

(Examples 1-7)
Various monomers shown in Table 1 and 90 g of water were weighed in a 200 ml four-necked flask. A stirring seal, a stirring rod, a reflux condenser, and a ceramic rubber were attached to each port. The system was placed in a constant temperature bath at 70 ° C., and the inside of the system was purged with nitrogen for 30 minutes while stirring at 200 rpm. Thereafter, 0.06 g of KPS, which is a polymerization initiator dissolved in water, was dissolved in 10 g of water and injected into the system with a syringe. At this time, the polymerization was started, and after 2 minutes, a predetermined amount of FeCl ₂ .4H ₂ O aqueous solution was injected using a syringe. The polymerization was carried out for 20 hours from the start of polymerization. In order to obtain an appropriate oxidizing power, 0.165 g of NH4OH was added during the polymerization.
The produced particles were purified by repeating centrifugation and redispersion four times with distilled water. At this time, centrifugation was performed at 20 ° C. and 13500 rpm. After centrifugation, the supernatant was discarded by decantation, distilled water was added, and redispersion was performed with a glass rod to obtain magnetic substance-encapsulated particles.

The descriptions in the table are as follows.
GMA: Glycidyl methacrylate EGDM: Ethylene glycol dimethacrylate AAm: Acrylamide PE-90: Polyethylene glycol methacrylate (n = 2)
PE-350: Polyethylene glycol methacrylate (n = 8)
NE-20:

(In the formula, X represents H.)

SE-20:

(In the formula, X represents SO ₄ NH _4. )

  With respect to the obtained magnetic substance-encapsulated particle dispersion, the particle dispersion state was visually observed. Further, the purified magnetic inclusion particles were diluted with water, deposited and fixed on a collodion film supported by a metal mesh, and the morphology of the particles was observed with a transmission electron microscope (TEM).

  In Example 1, since some agglomerates were observed and the dispersion stability in which particles settled with time was somewhat low, the particles were redispersed by ultrasonic treatment. On the other hand, in Examples 2 to 5 using polyethylene glycol methacrylate as a hydrophilic monomer and Examples 6 and 7 using a reactive emulsifier, no agglomerates were observed, and particles having high dispersion stability were obtained. It was. In particular, Examples 6 and 7 using reactive emulsifiers were found to have a small particle size and excellent dispersion stability. Moreover, it was observed that all the particles of Examples 1 to 7 contained a magnetic substance inside the particle, and the particle surface had a clean outline. FIG. 1 shows a TEM photograph (average particle diameter: magnetic inclusion particles 0.21 μm, magnetic substance 5 nm) of magnetic inclusion particles of Example 2.

  The produced magnetic substance-encapsulated particles (Examples 1 to 7) were taken in a small amount in a 1.5 ml microtube to confirm that they were attracted to the magnet, and diluted appropriately with distilled water to make a microtube stand with a magnet (manufactured by DYNAL). , MPC (registered trademark) _M), and it was visually confirmed that dispersed particles were attracted to the magnet. In particular, it was revealed that Examples 2 to 5 using polyethylene glycol methacrylate as the hydrophilic monomer had a larger magnetic force than other Examples. Table 2 shows the average particle size of the magnetic substance-encapsulated particles in each example.

(Example 8)
Various monomers and water shown below were weighed in a 200 ml four-necked flask.
AAm / GMA / EGDM / H2O = 0.15 / 2.835 / 0.015 / 90 (g)
A stirring seal, a stirring rod, a reflux condenser, and a ceramic rubber were attached to each port. The system was placed in a constant temperature bath at 70 ° C., and the inside of the system was purged with nitrogen for 30 minutes while stirring at 200 rpm. Thereafter, 0.06 g of KPS, which is a polymerization initiator dissolved in water, was dissolved in 10 g of water and injected into the system with a syringe. At this time, the polymerization was started, and after a predetermined time, an FeCl ₂ .4H ₂ O aqueous solution (0.165 g of FeCl ₂ .4H ₂ O was dissolved in 5 g of water) was injected using a syringe. NH ₄ OH / H ₂ O = 0.165 / 5 (g) was added to obtain an appropriate oxidizing power 1 minute after the start of polymerization, and polymerization was carried out for 2 hours.

  The produced particles were purified by repeating centrifugation and redispersion four times with distilled water. At this time, centrifugation was performed at 20 ° C. and 13500 rpm. After centrifugation, the supernatant was discarded by decantation, distilled water was added, and redispersion was performed with a glass rod to obtain magnetic substance-encapsulated particles.

Example 9
Magnetic substance-encapsulated particles were obtained in the same manner as in Example 8 except that the following substances were added at a predetermined time after the start of polymerization and the polymerization time was 3 hours.
30 minutes after the start of polymerization, NH ₄ OH / H ₂ O = 0.165 / 5 (g)
60 minutes after the start of polymerization KPS / H ₂ O = 0.165 / 5 (g)

(Example 10)
Magnetic substance-encapsulated particles were obtained in the same manner as in Example 8 except that the following substances were added at a predetermined time after the start of polymerization and the polymerization time was 3 hours.
60 minutes after the start of polymerization KPS / H ₂ O = 0.165 / 5 (g), FeCl ₂ .4H ₂ O / H ₂ O = 0.088 / 5 (g)
120 minutes after the start of polymerization, GMA = 0.5 (g), NH ₄ OH / H ₂ O = 0.165 / 5 (g)

(Example 11)
Magnetic substance-encapsulated particles were obtained in the same manner as in Example 8 except that the following substances were added at a predetermined time after the start of polymerization and the polymerization time was 4 hours.
1 minute after the start of polymerization NH ₄ OH / H ₂ O = 0.165 / 5 (g)
120 minutes after the start of polymerization KPS / H ₂ O = 0.165 / 5 (g)

The obtained magnetic substance-encapsulated particles (Examples 8 to 11) were observed for particle morphology by TEM.
Each of Examples 9 and 10 was found to contain more magnetic material than Example 8 and to have an increased particle size. Moreover, in Example 11, it was observed that the inside contains a magnetic material comparable to Examples 9 and 10, and the particle surface has a clean outline. From previous experiments, it was speculated that the addition of NH 4 OH in the early stage of particle growth is a factor inhibiting growth. On the other hand, post-addition of the polymerization initiator is confirmed to be an effective means that the polymerization rate is almost 100%, and secondary particles are not formed and the particle surface is covered with the polymer. It was done. Table 3 shows the average particle size of the magnetic substance-encapsulated particles in each example.

Furthermore, immunoassay particles were prepared from the magnetic substance-encapsulated particles obtained in Example 2, and immunoassay was performed.
(Preparation of immunoassay particles)
6 ml of a phosphate buffer (100 mmol / l, pH 7.5) was added to 30 mg of the magnetic substance-encapsulated particles obtained in Example 2, and centrifuged at 15000 rpm for 20 minutes. 1 ml of a solution obtained by dissolving anti-HBsAg monoclonal antibody in a phosphate buffer solution (100 mmol / l, pH 7.5) so as to have a concentration of 0.25 mg / ml is added to the resulting precipitate, and the mixture is stirred and mixed at room temperature for 20 hours. did. Thereafter, in order to remove the unreacted anti-HBsAg monoclonal antibody, centrifugation was performed at 15000 rpm for 20 minutes, and the resulting precipitate was suspended in 6 ml of a phosphate buffer (100 mmol / l, pH 7.5). Centrifugation was again performed at 15000 rpm for 20 minutes. Thereafter, the obtained precipitate is suspended in 6 ml of a solution obtained by dissolving bovine serum albumin in a phosphate buffer (100 mmol / l, pH 7.5) so as to have a concentration of 1% by weight, and stirred at room temperature for 1 hour. Then, blocking treatment was performed to obtain immunoassay particles in which the anti-HBsAg monoclonal antibody was bound to the magnetic particles.
Next, in order to store the obtained immunoassay particles in a refrigerated state, centrifugation was performed at 15000 rpm for 20 minutes, and the resulting precipitate was washed with a phosphate buffer solution (both with a bovine serum albumin concentration of 1% by weight). 100 mmol / l, pH 7.5) and suspended in 6 ml of a solution in which sodium azide was dissolved to a concentration of 0.01% by weight, and immediately stored in a refrigerator.

(Preparation of test piece)
A nitrocellulose membrane (SRHF, manufactured by Nihon Millipore) is cut into a width of 30 cm and a length of 6 cm, and a reaction different from that used in the above immunoassay particles at a site 3 cm from the upper end in the length direction (reaction site) A solution prepared by dissolving an anti-HBsAg monoclonal antibody having an epitope in Tris-HCl buffer (10 mmol / l, pH 7.4) so as to have a concentration of 2.0 mg / ml was applied linearly with a width of 0.7 mm. Thereafter, after drying at 37 ° C. for 2 hours, 1 in a solution obtained by dissolving bovine serum albumin (manufactured by Wako Pure Chemical Industries, Ltd.) in a phosphate buffer solution (100 mmol / l, pH 7.5) to a concentration of 1% by weight. It was immersed for a period of time and subjected to a blocking treatment. Further, after washing with a solution obtained by dissolving sodium laurylbenzenesulfonate in a phosphate buffer solution (100 mmol / l, pH 7.5) so as to have a concentration of 0.1% by weight, the mixture was washed at room temperature in a silica gel desiccator. The membrane was dried to obtain an anti-HBsAg monoclonal antibody-immobilized membrane.
The obtained anti-HBsAg monoclonal antibody-immobilized membrane was cut into a width of 5 mm, a water-absorbing filter paper (manufactured by Millipore Japan, Inc.) having a width of 5 mm and a length of 2 cm was stacked on the upper end in the length direction, and fixed with a transparent tape. It was.

(Immunity measurement)
The above immunoassay particles are dissolved in a phosphate buffer (100 mmol / l, pH 7.5) to a concentration of 0.1% by weight, and bovine serum albumin is dissolved to a concentration of 1% by weight. Further, a solution in which sodium azide was dissolved to a concentration of 0.01% by weight was prepared, and 20 μl of the solution was added to each well of a 96-well microplate (manufactured by Nargenunk International).
Next, the HBs antigen standard (50 IU / ml) was diluted with a phosphate buffer (100 mmol / l, pH 7.5) to a predetermined concentration, and 100 μl of each was added to the well and mixed. I stood inside and stood to stand.
After 30 minutes, when the test piece was taken out, magnetism corresponding to the HBs antigen concentration was confirmed at the reaction site, and it was shown to be useful for an immunoassay method using magnetism as a label.

4 is a TEM photograph of magnetic body-encapsulated particles of Example 2 (average particle diameter; magnetic body-encapsulated particles 0.21 μm, magnetic body 5 nm).

Claims (14)

  A magnetic substance-encapsulated particle comprising an organic polymer substance and a magnetic substance having an average particle diameter of 1 to 30 nm, wherein the magnetic substance is contained in a dispersed state therein.   2. The magnetic substance-encapsulated particle according to claim 1, wherein the magnetic substance is formed by oxidizing a metal ion inside the particle in a polymerization process for forming the particle.   3. The magnetic substance-encapsulated particle according to claim 2, wherein the metal ion is an iron ion.   4. The magnetic substance-encapsulated particle according to claim 1, 2 or 3, wherein the organic polymer substance comprises a polymer composed of an acrylic monomer as a main constituent component.   The magnetic substance-encapsulated particle according to claim 4, wherein the acrylic monomer is a monomer having a glycidyl group. 5. The magnetic substance-encapsulated particle according to claim 4, wherein the acrylic monomer is a monomer having a glycidyl group and polyethylene glycol (meth) acrylate represented by the following general formula.
_{CH 2 = CR-COO- (CH} 2 -CH 2 -O) n-H
(In the formula, R represents H or CH ₃ , and n represents an integer of 2 to 20)   7. The magnetic substance-encapsulated particle according to claim 1, wherein the average particle diameter is 0.05 to 1 [mu] m.   The magnetic substance-encapsulated particle according to claim 1, wherein the content of the magnetic substance is 0.1 to 40% by weight. A step of polymerizing a hydrophobic monomer and / or a hydrophilic monomer in an aqueous solvent to form particles;
The step of oxidizing the metal ions while incorporating the metal ions into the particles to form a magnetic material,
A method for producing a magnetic substance-encapsulated particle, wherein the step of forming the particle and the step of forming the magnetic body are simultaneously performed.   The method for producing magnetic substance-encapsulated particles according to claim 9, wherein the hydrophobic monomer is an acrylic monomer containing a glycidyl group. The method for producing magnetic body-encapsulated particles according to claim 9, wherein the hydrophilic monomer is polyethylene glycol (meth) acrylate represented by the following general formula.
_{CH 2 = CR-COO- (CH} 2 -CH 2 -O) n-H
(In the formula, R represents H or CH ₃ , and n represents an integer of 2 to 20)   The method for producing magnetic inclusion particles according to claim 9, 10 or 11, wherein a reactive emulsifier is added as a copolymerization monomer in the step of forming particles.   The method for producing magnetic substance-encapsulated particles according to claim 9, 10, 11, or 12, wherein a polymerization initiator is added later in the step of forming particles.   It is composed of an organic polymer substance and a magnetic substance having an average particle diameter of 1 to 30 nm, and an antigen or an antibody is adsorbed or bound to a magnetic substance-encapsulated particle containing the magnetic substance in a dispersed state therein. Particles for immunoassay.

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